The control of overt differentiation of mammary gland in vitro involves an interplay between polypeptide and steroid hormones. 1) There is absolute requirement of hydrocortisone for accumulation of these messages, specifically the accumulation of 42K casein mRNA in mammary tissue from adrenalectomized, virgin rat is almost 20x higher in the presence of exogenous hydrocortisone than in its absence. Accumulation of 25K casein mRNA is also totally dependent on the steroid. 2) Insulin is absolutely required for the expression of these milk protein genes and can be considered as a developmental hormone in the mammary system. Neither fetal calf serum nor Multiplication stimulating activity (MSA) or epidermal growth factor (EFG) can substitute insulin effect on differentiation, though these hormones can sustain mammary cell viability in culture.